Hi!
I will be performing transformation in E.coli Stbl3 strain followed by plasmid extraction. However, the Stbl3 strain is EndA+ strain of E. coli . Can someone please guide me with some alternatives to avoid degradation of my plasmid during or after extraction from transformed E.coli Stbl3 strain?
Note: Following plasmid extraction, the plasmid will be suspended and stored in TE buffer.
Regards,
Rajeshwari K.
Hi
Standard kits take this into consideration and have appropriate buffers for optimal extraction. You should be fine, don't worry about it.
So it very much does depend upon the kit as to whether EndA nuclease is removed or not. Some have an extra step of proteinase treatment, some introduce an extra wash step, etc. But often you do need extra precautions. Other kids however are fine as is.
However a trick we learned in my lab is that RNA is inhibitory to EndA nuclease. So if we do quick and dirty minipreps (not with a kit) for plasmid screening, then so long as you do NOT treat your samples with RNAse beforehand, you can still digest your mini preps fine and run on a gel. If the RNA blob at the bottom of the gel is annoying, we often would just have RNAse in our gel loading buffer and a few minutes treatment would suffice to clean up the RNA blob but still let you see clean bands.
But if you need really clean plasmid DNA for your downstream steps, then just be sure your plasmid prep kit is qualified to deal with EndA+ strains. Note that BL21 for example is also EndA+.
Thank you so much. I shall introduce an extra wash step and check Michael J. Benedik
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